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2017 Human H1 resolves R-loops and thereby enables progression of the DNA replication fork Shankar Parajuli Washington University School of Medicine in St. Louis

Daniel C. Teasley Washington University School of Medicine in St. Louis

Bhavna Murali Washington University School of Medicine in St. Louis

Jessica Jackson Saint Louis University

Alessandro Vindigni Saint Louis University

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Recommended Citation Parajuli, Shankar; Teasley, Daniel C.; Murali, Bhavna; Jackson, Jessica; Vindigni, Alessandro; and Stewart, Sheila A., ,"Human ribonuclease H1 resolves R-loops and thereby enables progression of the DNA replication fork." Journal of Biological Chemistry.292,37. 15216-15224. (2017). https://digitalcommons.wustl.edu/open_access_pubs/6278

This Open Access Publication is brought to you for free and open access by Digital Commons@Becker. It has been accepted for inclusion in Open Access Publications by an authorized administrator of Digital Commons@Becker. For more information, please contact [email protected]. Authors Shankar Parajuli, Daniel C. Teasley, Bhavna Murali, Jessica Jackson, Alessandro Vindigni, and Sheila A. Stewart

This open access publication is available at Digital Commons@Becker: https://digitalcommons.wustl.edu/open_access_pubs/6278 ARTICLE cro

Human ribonuclease H1 resolves R-loops and thereby enables progression of the DNA replication fork Received for publication, March 21, 2017, and in revised form, June 21, 2017 Published, Papers in Press, July 17, 2017, DOI 10.1074/jbc.M117.787473 Shankar Parajuli‡, Daniel C. Teasley‡, Bhavna Murali‡, Jessica Jackson§, Alessandro Vindigni§¶1, and Sheila A. Stewart‡¶ʈ**2 From the Departments of ‡Cell Biology and Physiology and ʈMedicine, ¶Siteman Cancer Center, and **Integrating Communications within the Cancer Environment (ICCE) Institute, Washington University School of Medicine, St. Louis, Missouri 63110 and the §Edward A. Doisy Department of Biochemistry and Molecular Biology, Saint Louis University School of Medicine, St. Louis, Missouri 63104 Edited by Patrick Sung

Faithful DNA replication is essential for genome stability. To the stalling and eventual collapse of replication forks (1). One

ensure accurate replication, numerous complex and redundant such structure is the RNA–DNA hybrid, commonly referred to Downloaded from replication and repair mechanisms function in tandem with the as an R-loop. Mapping studies have revealed that RNA–DNA core replication proteins to ensure DNA replication continues hybrid structures are present throughout the genome more fre- even when replication challenges are present that could impede quently than previously appreciated (2, 3). One source of progression of the replication fork. A unique topological chal- R-loops is transcription during which nascent RNA emanating lenge to the replication machinery is posed by RNA–DNA from RNA II hybridizes with its template DNA (4). http://www.jbc.org/ hybrids, commonly referred to as R-loops. Although R-loops Although these topological structures play a vital role in a num- play important roles in expression and recombination at ber of key processes including class switch recombination of immunoglobulin sites, their persistence is thought to interfere immunoglobulingenesandtranscriptiontermination,theirper- with DNA replication by slowing or impeding replication fork sistence or unscheduled formation and stabilization pose a sig- progression. Therefore, it is of interest to identify DNA-associ- nificant challenge to genome integrity (5, 6). Because DNA ated that help resolve replication-impeding R-loops. replication and transcription occur simultaneously at many at Washington University on October 26, 2017 Here, using DNA fiber analysis, we demonstrate that human regions of the genome, hybrids can form in front of the replica- ribonuclease H1 (RNH1) plays an important role in replication tion machinery and affect its progression. Indeed, these R-loops fork movement in the mammalian nucleus by resolving R-loops. can lead to increased DNA mutations, unwanted recombina- We found that RNH1 depletion results in accumulation of tion, and gross chromosomal aberrations (7). Thus, it is not RNA–DNA hybrids, slowing of replication forks, and increased surprising that a number of proteins inhibit the formation of DNA damage. Our data uncovered a role for RNH1 in global these structures or resolve them once they have formed (4). DNA replication in the mammalian nucleus. Because accumu- I, mRNA export, and splicing factors, for exam- lation of RNA–DNA hybrids is linked to various human cancers ple, play an active role in preventing R-loop formation (4, 8). and neurodegenerative disorders, our study raises the possibil- Conversely, the Senataxin (SETX)3 and Aquarius ity that replication fork progression might be impeded, adding (AQR) are tasked with resolving these structures to promote to increased genomic instability and contributing to disease. transcriptional termination and maintain genome stability, respectively (6, 9). In addition, DNA damage response factors such as breast cancer susceptibility factors (BRCA1 and High-fidelity DNA replication is paramount to the mainte- BRCA2) are also implicated in preventing R-loop accumulation nance of genome stability. Therefore, cells have evolved various and the ensuing DNA damage (10, 11). redundant mechanisms to resolve genotoxic challenges includ- Ribonuclease H1 (RNH1) is a specialized that can ing the presence of topological structures. If not resolved, these specifically resolve long RNA–DNA hybrids. A closely related structures can impede replication fork progression, leading to protein complex, ribonuclease H2 (RNH2), is adept at remov- ing single misincorporated ribonucleotides from DNA and is This work was supported by a Predoctoral Cancer Biology Pathway grant, critical for ongoing genomic stability (12). In yeast, RNH1 and Siteman Cancer Center/Barnes Jewish Hospital (to S. P.); National Institutes RNH2 function redundantly to facilitate efficient double strand of Health Molecular Oncology Training Grant T32CA113275 (to B. M.); National Institutes of Health Grant R01GM108648 (to A. V.); United States break repair during homologous recombination by assisting in Department of Defense Breast Cancer Research Program Breakthrough the unwinding of DNA strands and bind- Award BC151728 (to A. V.); National Institutes of Health Grant CA130919 (to S. A. S.); and an American Cancer Society research scholar award (to S. A. S.). The authors declare that they have no conflicts of interest with 3 The abbreviations used are: SETX, Senataxin; AQR, Aquarius; RNAH, ribonu- the contents of this article. The content is solely the responsibility of the clease H; CO, chromosomal orientation; siCtrl, control siRNA; siRNH1, authors and does not necessarily represent the official views of the RNH1-directed siRNA; DIP, DNA–RNA immunoprecipitation; maRTA, National Institutes of Health. microfluidic-assisted replication track analysis; CldU, 5-chloro-2Ј-deoxy- 1 To whom correspondence may be addressed. E-mail: [email protected]. uridine; IdU, 5-iodo-2Ј-deoxyuridine; BrdC, 5-bromo-2Ј-deoxycytidine; 2 To whom correspondence may be addressed. E-mail: sheila.stewart@ qRT, quantitative reverse transcription; ␥H2AX, histone H2AX phosphory- wustl.edu. lation at Ser-139; FAM, 6-fluorescein amidite.

15216 J. Biol. Chem. (2017) 292(37) 15216–15224 © 2017 by The American Society for Biochemistry and Molecular Biology, Inc. Published in the U.S.A. Human ribonuclease H1 facilitates replication fork movement ing (13). Ectopic expression of RNH1 in yeast is sufficient tion and abrupt excision. A second study suggested to minimize transcription-dependent hyper-recombination, that RNH1 plays an important role in resolving RNA–DNA pausing of the replication fork, and hydroxyurea sensitivity hybrids at the telomere (23). Because the leading strand-repli- (14–16). In mammalian cells, RNH1 has an established role in cated telomere is transcribed, RNA–DNA hybrids would be mitochondrial DNA replication, and its deletion is embryoni- expected to form on the leading strand. Thus, we examined the cally lethal, demonstrating that RNH2 cannot compensate in integrity of the leading strand telomere by performing chromo- this setting (17–20). RNH1 localizes to the mammalian nucleus somal orientation fluorescence in situ hybridization (CO- (21), and ectopic RNH1 expression is routinely exploited in FISH), which allows one to interrogate the leading versus mammalian cells to resolve RNA–DNA hybrids. Recently, lagging strand-replicated telomere. Surprisingly, CO-FISH RNH1 was shown to prevent unwanted recombination events analysis revealed no differences in the leading versus lagging at the telomere by resolving telomeric RNA–DNA hybrids in strand telomere in control versus shRNH1 cells (data not cells that utilize the alternative lengthening mechanism of shown). However, in the RNH1-depleted cells, we observed a telomere maintenance (22). Another recent study identified a significant increase in telomere free ends in which both leading link between DNA damage and the accumulation of RNA– and lagging strand were lost, a phenotype suggestive DNA hybrids at the telomere (23). However, whether RNH1 of DNA replication defects (26) (Fig. 1, C and D). These data plays a role in nuclear DNA replication outside of telomeres suggest that RNH1 assists the replication machinery by resolv- remains to be explored. ing RNA–DNA hybrids that could present a topological barrier Downloaded from Given the role of RNA–DNA hybrids in replication impair- to replication fork progression. ment and the ability of RNH1 to resolve such hybrids, we sought to determine whether RNH1 impacts genomic integrity Nuclear RNA–DNA hybrid levels increase upon RNH1 in the mammalian nucleus and if so how. We depleted RNH1 depletion from human cell lines and found that RNH1 depletion resulted http://www.jbc.org/ in increased RNA–DNA hybrids, DNA damage response, and To demonstrate that RNA–DNA hybrids were responsible slowing of DNA replication forks. Importantly, these pheno- for the DNA damage and telomere loss phenotype upon RNH1 types were dependent upon RNH1 activity, suggesting loss, we next measured the hybrid levels in the nucleus. We that the hybrids were responsible for these phenotypes. Our treated 293T cells with control (siCtrl) or RNH1-directed studies uncover a novel role of RNH1 in the mammalian (siRNH1) siRNAs and collected cells 48 h later. Transfection at Washington University on October 26, 2017 nucleus and extend its important function in nuclear DNA with siRNH1 led to a significant reduction in RNH1 mRNA replication. levels (2.5-fold) (Fig. 2A) and protein levels (3.5-fold) (Fig. 2B) compared with levels present in siCtrl cells. To measure the Results amount of RNA–DNA hybrids in control versus RNH1-de- RNH1 contributes to genome stability and preserves pleted cells, we next extracted nuclear DNA lysate and sub- telomere integrity jected it to DNA–RNA immunoprecipitation (DIP) using the well-characterized RNA–DNA hybrid antibody S9.6 (27). We Although RNH2 has well-ascribed functions in the mamma- conducted a genomic quantitative PCR on a well-characterized lian nucleus, the role of RNH1 has remained more obscure. hybrid-forming 5Ј pause site of ␤-actin gene as a readout of Because R-loops form throughout the genome and RNH1 can hybrids. As a control for specificity, we also pretreated lysates resolve R-loops that would pose barriers to the replication with recombinant RNaseH enzyme in vitro to degrade existing machinery, we hypothesized that RNH1 might play an impor- RNA–DNA hybrids in both control and depleted cells. As tant role in the nucleus and that its loss might perturb replica- tion fork progression and thus elicit a DNA damage response expected, pretreatment with an in vitro RNaseH enzyme led to (4). To test this hypothesis, we depleted RNH1 from normal, a 1.8-fold reduction of RNA–DNA hybrids in control and a checkpoint-competent RPE1 cells and measured the levels of 3.5-fold in RNH1-depleted cells, confirming the specificity of histone H2AX phosphorylation at Ser-139 (␥H2AX), a canon- the S9.6 antibody. Additionally, immunoprecipitation with an ical marker of the DNA damage response (24), in control versus IgG control antibody failed to precipitate RNA–DNA hybrids, RNH1-depleted cells. Following RNH1 depletion, we observed indicating that the signals we measured were bona fide RNA– increased levels of ␥H2AX, demonstrating that RNH1 deple- DNA hybrids. Analysis of immunoprecipitations from RNH1- tion induces a DNA damage response (Fig. 1, A and B). These depleted cells revealed a significant 2-fold increase in the data suggest that RNH1 plays an important role in preserving nuclear RNA–DNA hybrids compared with those in control genome stability. cells (Fig. 2C). To further corroborate these findings, we also To further interrogate the function of RNH1, we first focused utilized the S9.6 antibody and carried out immunofluorescence our attention to telomeres, chromosomal ends that contain on RPE1 cells. As expected, RNH1-depleted cells showed RNA–DNA hybrids (25). Recent work demonstrates that in increased levels of RNA–DNA hybrids as represented by ele- cells utilizing the alternative lengthening of telomere mecha- vated S9.6 signal in the nucleus compared with that in the con- nism, which maintains telomere length independently of trol cells (Fig. 2, D and E). Together, these data demonstrate , RNH1 associates with telomeres and regulates the that RNH1 depletion can lead to a significant increase in RNA– levels of telomeric RNA–DNA hybrids to prevent telomere loss DNA hybrids and that this increase correlates with increased (22). In these cells, depletion of RNH1 led to hybrid accumula- DNA damage and telomere loss.

J. Biol. Chem. (2017) 292(37) 15216–15224 15217 Human ribonuclease H1 facilitates replication fork movement Downloaded from http://www.jbc.org/ at Washington University on October 26, 2017

Figure 1. RNH1 contributes to genome stability and preserves telomere integrity. A, Western analysis of RNH1 expression and ␥H2AX in control (shCtrl) and RNH1-depleted RPE1 cells (shRNH1). Bleomycin-treated cells (Bleo) served as a positive control for ␥H2AX. ␣-Tubulin is shown as a loading control. Molecular mass in kilodaltons is marked to the right for reference. B, quantification of ␥H2AX intensity in shCtrl and shRNH1 cells from the Western blot in A. C, representative metaphase processed with CO-FISH from shCtrl or shRNH1 RPE1 cells. Leading strand-replicated telomeres are green, and lagging strand-replicated telomeres are red. Regions marked by white asterisks are magnified; white arrows indicate telomere free ends (TFE) in magnified images. D, representative quantification of telomere loss in shCtrl and shRNH1 RPE1 cells. A minimum of 700 metaphase chromosomes were analyzed. p values were computed using a two-tailed Student’s t test (*, p Ͻ 0.05). Error bars represent S.E.

RNH1 depletion results in replication fork slowing and uring the lengths of these IdU tracks, we found that RNH1- increased termination and stalling depleted cells had an average IdU track length of 9.3 Ϯ 0.3 ␮ Ϯ ␮ Given the increased RNA–DNA hybrids, DNA damage, and m, whereas that in the control cells was 14.5 0.4 m, loss of both telomeric ends, indicative of a replication defect indicating that the replication forks moved significantly following RNH1 depletion, we hypothesized that RNA–DNA slower in RNH1-depleted cells compared with control cells hybrids pose barriers to DNA replication forks. This hypothesis (Fig. 3C). Given the increase in RNA–DNA hybrids associ- was supported by previous studies showing that the removal ated with RNH1 loss, these data suggest that RNH1 facili- of RNA–DNA hybrids by ectopically expressed RNH1 can tates efficient DNA replication by clearing RNA–DNA directly affect replication fork movement in yeast (28). To test hybrids that would otherwise impede replication fork pro- this hypothesis, we used microfluidic-assisted replication track gression during S phase. analysis (maRTA) to directly measure replication fork progres- To understand how the loss of RNH1 perturbed replication sion in RPE1 cells depleted of RNH1 (29, 30). RPE1 cells were dynamics, we next asked whether other replication parameters transduced with siRNAs, and cells were collected for West- including termination, stalling, and origin firing were affected ern blot analysis 48 h later. As expected, RNH1 depletion upon RNH1 depletion. Premature termination and stalling resulted in significant DNA damage as evidenced by in- events correspond to CldU (red)-only tracks (Fig. 3B). RNH1- creased ␥H2AX (Fig. 3A). In parallel, we carried out depleted cells showed a significant 1.4-fold increase in termina- maRTA by plating RNH1-depleted or control cells and label- tion and/or stalling events compared with control cells (Fig. ing them with the nucleotide analogs CldU (red) and IdU 3D). Next, we analyzed the impact of RNH1 depletion on origin (green) sequentially for 30 min each to allow us to follow firing to address a possibility that slower fork progression trig- replication fork movement (Fig. 3B). To restrict our analysis gers the S phase checkpoint and increases the frequency of to progressing replication forks, we measured IdU tracks origin firing as reported previously (31). To measure origin fir- that were directly preceded by a CldU track (Fig. 3B). Meas- ing, the incidence of tracks with either green-only (IdU) color

15218 J. Biol. Chem. (2017) 292(37) 15216–15224 Human ribonuclease H1 facilitates replication fork movement Downloaded from http://www.jbc.org/

Figure 2. Nuclear RNA–DNA hybrid levels increase upon RNH1 depletion. A, qRT-PCR analysis of RNH1 mRNA in 293 T cells transfected with siCtrl or siRNH1. ⌬⌬ Expression levels were calculated using the Ct method and normalized relative to GAPDH expression. B, Western analysis of RNH1 expression in siCtrl and RNH1-depleted 293T cells (siRNH1). ␣-Tubulin is shown as a loading control. Molecular mass in kilodaltons is marked to the right for reference. C, quantification of the DIP signal shown as a percentage of input in siCtrl and siRNH1 293T cells. Pretreatment of lysate with in vitro RNaseH (RNAH) enzyme serves as a control for RNA–DNA hybrids. IgG is a nonspecific antibody, whereas S9.6 is an RNA–DNA hybrid-specific antibody. Analysis of three technical repeats from a Ͻ

representative experiment is shown. p values were computed using a three-way analysis of variance with Sidak multiple comparison test (*, p 0.05). Error bars at Washington University on October 26, 2017 represent S.E. D, representative images of S9.6 immunofluorescence on RPE1 control (siCtrl) and RNH1-depleted (siRNH1) cells. Blue staining marks the nuclei, and red is S9.6 signal (RNA–DNA hybrids). E, quantification of S9.6 signal (raw integrated density) (arbitrary units) for siCtrl and siRNH1 cells. Shown is one of three independent experiments where a minimum of 80 nuclei were analyzed per sample. p values were computed using a non-parametric Mann-Whitney test (*, p Ͻ 0.05). Error bars represent S.E.

or red flanked by green on both sides was analyzed (Fig. 3B). expressed protein. We observed that RNH1 depletion using a However, no significant differences in origin firing were 3Ј-UTR siRNA was comparable with that of a previously used observed between RNH1-depleted and control cells (Fig. coding sequence targeting siRNA (Fig. 4B). We also observed 3E). Collectively, these results indicate that RNH1 plays an robust expression of our ectopically tagged RNH1 proteins and important role in assisting fork movement during DNA rep- significant depletion of endogenous RNH1 levels (Fig. 4B). lication. We suggest that RNH1 does this by resolving RNA– Next, using maRTA, we again measured replication fork move- DNA hybrids that pose barriers to a progressing replication ment and found that ectopic expression of WT RNH1 restored fork. Furthermore, our data provide the first evidence that fork movement to the levels observed in siCtrl cells (Fig. 4C). RNH1 plays a role in global DNA replication in the mamma- Indeed, although RNH1-depleted forks moved an IdU length of lian nucleus. 8 ␮m, ectopic expression of WT RNH1 increased this to 12 ␮m, levels we observed in siCtrl cells. In contrast, ectopic expression Nuclease activity of RNH1 is required for efficient replication of the catalytically dead D145N allele of RNH1 failed to rescue fork movement the replication fork movement defect (fork movement was 7.9 To further characterize the role of RNH1 in DNA replication, ␮m, nearly identical to that observed in RNH1-depleted cells). we tested whether the nuclease function of RNH1 was required Together, these findings demonstrated that the nuclease activ- for this activity. To do this, we created a series of RPE1 cell lines ity of RNH1 is required for the unperturbed movement of rep- ectopically expressing either a GFP-tagged wild-type (WT) or a lication forks in mammalian cells. Similarly, we also measured well-characterized GFP-tagged nuclease-dead (D145N) form of fork termination and stalling events upon ectopic expression of RNH1 (32, 33). To establish a direct role in the nucleus, these WT and D145N alleles in RNH1-depleted cells. As expected, RNH1 constructs lacked the mitochondrial targeting sequence both events were reversed by ectopic expression of WT RNH1 present in the endogenous gene, thereby restricting their but not the nuclease-dead allele, thereby reiterating the impor- expression to the nucleus. We confirmed the nuclear local- tance of the nuclease function of RNH1 in the fidelity of repli- ization of ectopically expressed proteins by visualizing GFP cation fork progression (Fig. 4D). Neither WT nor the D145N expression only in the nucleus (Fig. 4A). These stable RPE1 cells allele of RNH1 affected the levels of origin firing in RNH1- were transfected with an siRNH1 directed toward the 3Ј-un- depleted cells (Fig. 4E). These data suggest that the nuclease translated region (3Ј-UTR) that did not target the ectopically activity of RNH1 is required for resolution of RNA–DNA

J. Biol. Chem. (2017) 292(37) 15216–15224 15219 Human ribonuclease H1 facilitates replication fork movement

A B siCtrl siRNH1

30 min CldU 30 min ldU γ H2AX 20 IF 15

Cultured Cells RNH1 37 25 Ongoing Fork Fork Termination/ Stall 75 Origin Firing α-Tubulin 50 10 uumm siCtrl C 50 * siRNH1

40 D E

80 Downloaded from 50 30 ns 60 * 40

30

20 http://www.jbc.org/ 40

IdU Track Length (um) Length Track IdU 20 10

20 Origin% of Firing 10 % of Terminated or Stalled or Forks Terminated % of at Washington University on October 26, 2017 0 0 0 siCtrl siRNH1 siCtrl siRNH1 siCtrl siRNH1 Figure 3. RNH1 depletion results in replication fork slowing and increased termination and stalling. A, Western analysis of RNH1 expression and ␥H2AX in RPE1 cells transfected with siCtrl and siRNH1. ␣-Tubulin is shown as a loading control. Molecular mass in kilodaltons is marked to the right for reference. B, schematics showing labeling of cells for maRTA. Transfected RPE1 cells were labeled with base analogs CldU and IdU for 30 min each and subjected to the maRTA protocol and DNA visualization in red (CldU) and green (IdU) by immunofluorescence (IF). Ongoing forks were marked by a red track (IdU) followed by green (CldU); terminated and/or stalled were red-only tracks; origin firings were both green-only and red flanking green on either side. Representative DNA tracks for siCtrl and siRNH1 samples are shown. C, a representative quantification of three independent biological experiments showing the IdU track length (␮m) preceded by a CldU track. Analysis included a minimum of 260 two-color DNA tracks (moving fork) isolated from siCtrl and siRNH1 cells each. p values were computed using a two-tailed Student’s t test (*, p Ͻ 0.05). Error bars represent S.E. D, quantification of percentage of termination and stalling events in DNA isolated from siCtrl and siRNH1 samples. Means from three independent experiments were analyzed, and each analysis included between 210 and 260 DNA tracks per sample. p values were computed using a two-tailed Student’s t test (*, p Ͻ 0.05). Error bars represent S.E. E, quantification of the percentage of origin firings in DNA isolated from siCtrl and siRNH1 samples. The graph represents combined means from three independent experiments that included between 275 and 350 DNA tracks per sample. p values were computed using a two-tailed Student’s t test (ns, p Ͼ 0.05). Error bars represent S.E.

hybrids and therefore efficient movement of replication forks yeast have all been shown to resolve RNA–DNA hybrids (9, during nuclear DNA replication. 34–36). Here, we add RNH1 to a growing list of proteins by showing that endogenous RNH1 is required to similarly remove Discussion RNA–DNA hybrids and that if these hybrids are not removed Our study establishes a role for RNH1 in genomic DNA rep- replication is significantly impacted. Furthermore, our work lication. Indeed, we illustrate for the first time that RNH1 demonstrates that these other proteins are unable to compen- nuclease activity is required for efficient fork movement during sate for loss of RNH1 in replication fork progression. However, nuclear DNA replication. Furthermore, we have established a how RNH1 is regulated in the nucleus and how its activity is correlation between the accumulation of RNA–DNA hybrids coordinated with the replication machinery remain unclear. A and replication defects observed upon RNH1 depletion. Taken recent study from Nguyen et al. (37) elegantly demonstrated together, we propose a model wherein RNH1 resolves RNA– that replication protein A can interact with RNH1 and stimu- DNA hybrids to assist the replication machinery in its uninter- late its activity, raising the possibility that RNH1 is tightly reg- rupted movement during DNA replication. ulated by the DNA replication and repair machinery. The unscheduled formation and stabilization of RNA–DNA Given that RNH1 loss elicits replication defects such as fork hybrids have been postulated to be detrimental to the replica- slowing, termination, and fork stalling, it will be critical to tion machinery. The importance of resolving these structures is determine how this impacts checkpoint activation and cell probably best underscored by the multitude of proteins that act cycle progression. Furthermore, understanding the fate of on RNA–DNA structures. Indeed, helicases such as SETX, accumulated RNA–DNA hybrids upon RNH1 depletion is AQR, and DHX9 in mammalian cells and Sen1 and PIF1 in another interesting avenue worth pursuing. As previously

15220 J. Biol. Chem. (2017) 292(37) 15216–15224 Human ribonuclease H1 facilitates replication fork movement Downloaded from http://www.jbc.org/ at Washington University on October 26, 2017

Figure 4. Nuclease activity of RNH1 is required for efficient replication fork movement. A, representative images verifying the nuclear localization (green) of ectopically expressed RNH1 in 293T cells transfected with a GFP-tagged WT RNH1 or nuclease-dead (D145N) allele. B, Western analysis of RNH1 expression (endogenous and ectopic) in RPE1 cells transfected with siCtrl or siRNH1 with or without ectopic expression of either GFP-tagged wild-type (siRNH1ϩ WT) or nuclease-dead (siRNH1ϩ D145N) RNH1. ␣-Tubulin is shown as a loading control. Molecular mass in kilodaltons is marked to the right for reference. C, a representative quantification of three independent biological experiments showing the IdU track length (␮m) preceded by a CldU track. Analysis included 265–280 two-color DNA tracks (ongoing fork) isolated from each of the four samples. p values were computed using a one-way analysis of variance with Bonferroni multiple comparison test (*, p Ͻ 0.05; ns, p Ͼ 0.05). Error bars represent S.E. D, quantification of percentage of termination and stalling events in isolated DNA from all four samples. Means from three independent experiments were analyzed, and each analysis included between 240 and 350 DNA tracks per sample. Error bars represent S.E. E, quantification of the percentage of origin firings in DNA isolated from all four samples. The graph shown represents combined means from three independent experiments that included between 225 and 250 DNA tracks per sample. Error bars represent S.E. noted, RNA–DNA hybrids arising from different sources can The study of R-loops and their resolution have sparked be processed via separate mechanisms (9). For example, those more attention in recent years due to the fact that R-loops involved in class switch recombination are not processed via are associated with a number of diseases including cancer nucleotide excision repair, whereas those arising from loss of and several neurodegenerative disorders (38). This under- some RNA processing factors or camptothecin treatment are scores a need for understanding these structures and their processed by nucleotide excision repair. It is also worth evalu- origins, stabilization, and resolution along with their impact ating whether redundant and helicases including on cellular processes. By revealing the function of RNH1 in RNH2, SETX, and AQR could rescue the effects of RNH1 R-loop resolution in the nucleus, our study adds to the diver- loss. sity of mechanisms targeting such structures. Furthermore,

J. Biol. Chem. (2017) 292(37) 15216–15224 15221 Human ribonuclease H1 facilitates replication fork movement

our study identifies a previously unknown function of RNH1 used: mouse monoclonal anti-RNase H1 (H00246243-M01, in nuclear DNA replication. Together, our work broadens Novus Biologicals, Littleton, CO), rat monoclonal anti-tubulin the understanding of RNA–DNA structures and places (NB600-506, Novus Biologicals), and mouse monoclonal anti- RNH1 as a novel mechanism to resolve those structures and phospho(Ser-139) H2AX (05-636, Millipore). assist in nuclear DNA replication. Metaphase preparation Experimental procedures Metaphase chromosomes were prepared as described previ- Cell culture ously (42). Briefly, cultured RPE1 cells were treated with 0.5 ␮g/ml Colcemid (Sigma) for 6 h. Arrested metaphase cells were Cells were cultured at 37 °C in 5% carbon dioxide and atmo- collected by mitotic shake-off, treated with 75 mM potassium spheric oxygen as reported previously (39). 293T cells were chloride, and fixed in 3:1 solution of methanol and acetic acid. obtained from Dr. Robert Weinberg (Massachusetts Institute Chromosomes were spread by dropping onto glass slides. For of Technology) and cultured in high-glucose Dulbecco’s mod- analysis via CO-FISH, 0.3 ␮g/ml BrdU (Sigma) and 0.1 ␮g/ml ified Eagle’s medium (DMEM) containing 10% heat-inactivated BrdC (MP Biomedicals, Santa Ana, CA) were added to the cul- fetal bovine serum (FBS) and 1% penicillin/streptomycin tured medium 18 h prior to collection of the cells. (Sigma-Aldrich). RPE1 cells were obtained from ATCC and cultured in DMEM/Nutrient Mixture F-12 containing 7.5% CO-FISH heat-inactivated FBS and 1% penicillin/streptomycin. Downloaded from CO-FISH was performed as described previously with mod- siRNA transfection ifications (43). Briefly, spread metaphase chromosomes were aged at 65 °C for 18 h. Aged chromosomes were rehydrated in siRNA transfection was performed using Invitrogen’s PBS, treated with 100 ␮g/ml RNase at 37 °C for 10 min, and Lipofectamine RNAiMAX reagent (Thermo Fisher Scien-

refixed in 4% paraformaldehyde at room temperature for 10 http://www.jbc.org/ tific) according to the manufacturer’s protocol. siRNAs used min. Fixed chromosomes were UV-sensitized in 0.5 ␮g/ml were siCtrl (catalog number 4390843) and siRNH1 (catalog Hoechst 33258 (Sigma) in 2ϫ SSC at room temperature for number 4390824, ID s48356) from Life Technologies or siRNA 15 min and exposed to 365 nm UV light for 1 h using a UV Ј directed to the 3 -UTR of RNH1 (hs.Ri.RNASEH1.13.1) from cross-linker (Vilber-Lourmat, Marne-la-Vallée, France). Exposed Integrated DNA Technologies. chromosomes were digested with 3 units/␮l III (Promega, Madison, WI) at room temperature for 15 min, at Washington University on October 26, 2017 Virus production, infections, and stable cell lines denatured in 70% formamide in 2ϫ SSC, and dehydrated in Lentiviral production and transductions were carried out as cold ethanol before hybridization. Chromosomes were hybrid- reported previously (40). Briefly, 293T cells were transduced ized first using a 0.03 ␮g/ml concentration of a leading strand with pLKO.1-puro plasmid carrying an shRNA using TransIT- telomere PNA probe (FAM-(TTAGGG)3) followed by a 0.03 LT1 reagent (Mirus Bio, Madison, WI) and a mixture con- ␮g/ml concentration of a lagging strand PNA probe (Cy3- taining 8:1 ratio of pHR-CMV-8.2 R packaging plasmid and (CCCTAA)3) (both probes from PNA Bio, Thousand Oaks, pCMV-VSV-G. Supernatant containing virus was collected CA). Hybridized chromosomes were mounted using ProLong 48 h post-transfection and filtered through a 0.45-␮m PVDF Gold (Life Technologies) with 125 ng/ml DAPI. membrane. RPE1 cells were infected for 4 h each on 2 consec- utive days in the presence of 8 ␮g/ml protamine sulfate (Sigma). maRTA Following infection, transduced cells were selected with 15 maRTA was conducted as described previously (29, 44). ␮g/ml puromycin sulfate. Stable RPE1 cell lines were prepared Briefly, asynchronous RPE1 cells were labeled for 30 min each by using either a GFP-tagged D145N RNH1 construct (a gen- with 50 ␮M CldU and 50 ␮M IdU with two PBS washes in erous gift from Dr. Marteijn) (32) or its wild-type version (mod- between. Labeled cells were collected and embedded in agarose ified from D145N construct using site-directed mutagenesis; plugs for lysis and DNA extraction. DNA was subsequently Agilent Technologies). stretched, denatured, and subjected to immunostaining. Anti- bodies used were rat anti-CldU/BrdU (Abcam, ab6326), mouse Western blot analysis anti-IdU/BrdU (BD Biosciences, 347580), goat anti-rat Alexa Western blot analysis was carried out as described previously Fluor 594 (Invitrogen, A11007), and goat anti-mouse Alexa with modifications (41). Briefly, cells were washed with PBS and Fluor 488 (Invitrogen, A11001). lysed in radioimmunoprecipitation assay buffer (150 mM NaCl, 50 mM Tris-HCl, pH 8.0, 1.0% Nonidet P-40, 0.5% sodium Fluorescence imaging deoxycholate, and 0.1% SDS) supplemented with 1 mM sodium Metaphase chromosomes from CO-FISH and labeled DNA orthovanadate, 1 mM sodium fluoride, 1 mM microcystin LR, 2 tracks from maRTA were imaged on a Nikon 90i epifluores- mM phenylmethylsulfonyl fluoride, protease inhibitor mixture cence microscope using a 100ϫ 1.40 numerical aperture Plan (Sigma), and inhibitor mixture set I (EMD Milli- Apo VC objective (Nikon Instruments, Melville, NY) with Car- pore, Billerica, MA). Following sonication and centrifugation, gille Type LDF immersion oil (Cargille Sacher Laboratories, supernatant lysate was quantified using a protein assay (Bio- Cedar Grove, NJ). Images were captured using a CoolSnap HQ2 Rad). Lysates were subjected to SDS-PAGE and transferred to charge-coupled device camera (Photometrics, Tucson, AZ) and PVDF membranes for blotting. The following antibodies were deconvoluted with a blind algorithm using NISElements AR

15222 J. Biol. Chem. (2017) 292(37) 15216–15224 Human ribonuclease H1 facilitates replication fork movement

(Nikon Instruments) prior to quantification. RPE1 cells stably gies) and GAPDH (Hs.PT.39a.22214836, Integrated DNA expressing GFP-tagged WT and D145N RNH1 were visualized Technologies). and captured without any staining. DIP Author contributions—Experiments were designed by S. A. S., A. V., S. P., and D. C. T. Experiments were performed by S. P., D. C. T., and DIP was performed as described previously with modifica- J. J. Data were analyzed by S. A. S., A. V., B. M., and S. P. The manu- tions (6). Briefly, 293T cells were pelleted and resuspended in script was written by S. A. S., A. V., and S. P. DIP lysis buffer (0.5% Nonidet P-40, 85 mM potassium chloride, and5mM PIPES). Following centrifugation, pelleted nuclei Acknowledgments—We thank Kevin C. Flanagan for helpful com- were lysed in DIP nuclear lysis buffer (1% sodium dodecyl sul- ments and Sandra Crocker and Shashikant Kulkarni for examination fate, 25 mM Tris-HCl, pH 8, and 5 mM EDTA), sheared, and of chromosomes. We also thank Zhongsheng You for antibodies. digested with two sequential rounds of 100 ␮g of proteinase K for 1.5 h each at 55 °C. DNA was phenol/chloroform-extracted and ethanol-precipitated (45) at which point one-half was sub- References jected to an overnight digestion with recombinant ribonuclease 1. O’Driscoll, M. (2017) The pathological consequences of impaired genome H (Roche Applied Science). Samples were then diluted in DIP integrity in humans; disorders of the DNA replication machinery. 241, dilution buffer (1.1% Triton X-100, 0.01% sodium dodecyl sul- J. Pathol. 192–207 2. Ginno, P. A., Lim, Y. W., Lott, P. L., Korf, I., and Chédin, F. (2013) GC skew Downloaded from fate, 1.2 mM EDTA, 16.7 mM Tris-HCl, pH 8, and 166.5 mM at the 5Ј and 3Ј ends of human links R-loop formation to epigenetic sodium chloride) and sonicated to generate ϳ200-bp-long regulation and transcription termination. Genome Res. 23, 1590–1600 DNA fragments. Resulting DNA was quantified using the 3. Wongsurawat, T., Jenjaroenpun, P., Kwoh, C. K., and Kuznetsov, V. (2012) PicoGreen assay following the manufacturer’s protocol (Life Quantitative model of R-loop forming structures reveals a novel level of Technologies). 10 ␮g of DNA was immunoprecipitated over- RNA-DNA interactome complexity. Nucleic Acids Res. 40, e16 ␮ 4. Santos-Pereira, J. M., and Aguilera, A. (2015) R loops: new modulators of http://www.jbc.org/ night with 10 g of S9.6 antibody or mouse IgG. Antibody– genome dynamics and function. Nat. Rev. Genet. 16, 583–597 DNA complexes were captured by using Protein A magnetic 5. Yu, K., Chedin, F., Hsieh, C.-L., Wilson, T. E., and Lieber, M. R. (2003) beads (Life Technologies) after equilibration in DIP dilution R-loops at immunoglobulin class switch regions in the chromosomes of buffer. After extensive washing, antibody–DNA complexes stimulated B cells. Nat. Immunol. 4, 442–451 were eluted from the beads and treated with proteinase K fol- 6. Skourti-Stathaki, K., Proudfoot, N. J., and Gromak, N. (2011) Human lowed by recovery using PCR clean-up columns (Qiagen, senataxin resolves RNA/DNA hybrids formed at transcriptional pause at Washington University on October 26, 2017 sites to promote Xrn2-dependent termination. Mol. Cell 42, 794–805 Venlo, Netherlands). 7. Aguilera, A., and García-Muse, T. (2012) R loops: from transcription by- S9.6 immunofluorescence products to threats to genome stability. Mol. Cell 46, 115–124 8. Li, M., Pokharel, S., Wang, J.-T., Xu, X., and Liu, Y. (2015) RECQ5-depen- S9.6 immunofluorescence was performed as described pre- dent SUMOylation of DNA topoisomerase I prevents transcription-asso- viously (9). Briefly, RPE1 cells transfected with siCtrl or siRNH1 ciated genome instability. Nat. Commun. 6, 6720 were fixed with ice-cold methanol for 5 min at Ϫ20 °C. Fixed 9. Sollier, J., Stork, C. T., García-Rubio, M. L., Paulsen, R. 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15224 J. Biol. Chem. (2017) 292(37) 15216–15224 Human ribonuclease H1 resolves R-loops and thereby enables progression of the DNA replication fork Shankar Parajuli, Daniel C. Teasley, Bhavna Murali, Jessica Jackson, Alessandro Vindigni and Sheila A. Stewart J. Biol. Chem. 2017, 292:15216-15224. doi: 10.1074/jbc.M117.787473 originally published online July 17, 2017

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